The ability of the body to arrest the flow of blood following vascular injury is paramount to continued survival. The process by which this occurs is termed haemostasis and is accomplished by the process of blood coagulation leading to formation of a blood clot or thrombosis. A blood clot consists of a plug of platelets enmeshed in a network of insoluble fibrin particles. Whilst formation of the clot is essential, the persistence of such clots would be dangerous to the body. Thus, in order to minimize damage to the body after the clotting process has served its purpose, healthy cells surrounding the clot release plasmin to digest fibrin, therefore dissolving the clot. However, thrombosis is one of the leading causes of death worldwide due to the flow of blood to vital organs and tissues being blocked by blood clots. Thrombosis may occur anywhere within the circulatory system, however it can be especially life threatening when this occurs in the lower body, heart, lungs or brain resulting in deep vein thrombosis, acute myocardial infarction, pulmonary embolism and acute isechemic stroke.
Two pathways or coagulation cascades lead to the formation of a clot, known as the intrinsic and extrinsic pathways. These two pathways are initiated by distinct mechanisms but converge along a common pathway. Clot formation in response to an abnormal vessel wall in the absence of tissue injury is the result of the intrinsic pathway and clot formation in response to tissue injury is the result of the extrinsic pathway. The coagulation cascades are very complex and involve a number of different proteins known as clotting factors.
People who suffer from cardiac or vascular diseases and patients that have undergone surgical procedures are at risk of developing blood clots that may result in life-threatening clinical conditions. Such people are often treated with blood-thinning or anticoagulant drugs. However, the amount of anticoagulant in the bloodstream must be maintained at the proper level; too little may result in unwanted clotting whilst too much can result in haemorrhaging. As a result routine coagulation screening tests have been developed in order to evaluate the coagulation status of blood or plasma.
A useful measure of coagulation is the so called prothrombin time (PT) test. The PT test was first developed in 1935 and measures the tissue factor-induced coagulation time of blood or plasma. This can provide an assessment of the extrinsic coagulation pathway and is sensitive to factors I, II, V, VII and X. The test is performed by adding a clotting agent such as thromboplastin and Ca2+ to a patient sample and measuring the time for clot formation. Portable coagulation monitors such as the CoaguChek® Plus coagulation meter have been developed which measure prothrombin time using non-anticoagulated capillary whole blood from a fingerstick or lancing device. Such monitors have been shown to be a valuable tool for patients on long-term oral anti-coagulation therapy.
However, the traditional expression of PT test results is inadequate for international comparison because the values depend upon the nature of the thromboplastin used. This has lead to the adoption of the Internationalised Normalised Ratio or INR as a way of expressing prothrombin time, where:INR=(PT ratio)ISIwhere ISI is the International Sensitivity Index andPT ratio=Patient's PT/ Mean Normal PT
The ISI is derived from the calibration line of the value of PT for a number of samples, obtained using a particular thromboplastin versus the World Health Organisation (WHO) international reference preparation for thromboplastin (human combined 67/40). A particular value of ISI, which takes into account the particular method and type of thromboplastin used, is assigned to each PT system, whereby each PT ratio can be translated into a standardized ratio. By employing INR, patients should be able to maintain a satisfactory level of coagulation which is independent of the PT system used.
Another method of measurement of coagulation in either blood or plasma is the Activated Partial Thromboplastin Time Test (APTT). This test is a measure of the time of coagulation that occurs when the intrinsic pathway is activated. This is achieved by the addition of an activator (kaolin) to the sample in the presence of calcium ions and phospholipid (partial thromboplastin). APTT is used to evaluate the intrinsic coagulation pathway which includes the factors I, II, V, VIII, IX, X, XI and XII. Formation of complexes on the surface of the phospholipid enables prothrombin to be converted into thrombin, which results in clot formation.
APTT is used as a routine test for monitoring heparin therapy during surgical procedures, as a preoperative screening test for bleeding tendencies and to assess the overall competence of the patient's coagulation system. This test is commonly carried out in the central laboratory.
Activated Clotting Time Test (ACT)
This test resembles the APTT test and is used to monitor a patient's coagulation status during procedures that involve the dosing of high amounts of heparin, such as percutaneous transluminal coronary angioplasty (PCTA) and cardiopulmonary bypass surgery. The ACT test is considered as one of the best laboratory tests for the control of heparin therapy, both for patients undergoing treatment for thromboembolic disease and for those on extra-corporeal circulation. For those patients taking heparin, prolongation of the ACT is directly proportional to the concentration of heparin in blood. Monotoring is important and underdosing or overdosing of heparin may result respectively in pathological thrombus formation or serious hemorrhagic conditions.
The original ACT test utilized a glass tube with a celite activator and it was necessary to invert the blood tube every 15–30 seconds so as to continually reexpose the blood sample to large amounts of glass. The MAX-ACT™ test has been developed by Helena Laboratories which overcomes the need to invert the tube yet at the same time providing a large exposure to glass by the use of additional glass beads.
Thrombin Time Test (TT)
This test measures the rate of formation of a fibrin clot in plasma by the action of thrombin on fibrinogen, compared to a normal plasma control. The test is performed by adding a standard amount of thrombin to a patient's plasma that has been deprived of platelets and measuring the time for a clot to form. It has been used in the diagnosis of disseminated intravascular coagulation and liver disease and is generally performed in the central laboratory.
Other Tests
Clotting assays have been developed which target specific factors such as factor VIIIa that is indicative of factor IX deficiency. Another example is an assay for factor VIII, which constitutes a test for haemophilia. Other tests include assays to measure the levels of activation peptide factor IXa, antithrombin, protein C and protein S.
Immunochemical assays have also been developed to identify and measure the various markers of coagulation and thrombosis.
Various instruments have developed for use in the laboratory and as POC. In addition to this, devices have been developed which allow the patients to home-monitor their blood coagulation. This is especially useful for patients who are on long-term anticoagulation therapy, such as warfarin.
Various techniques are employed to measure blood coagulation, as exemplified below.
U.S. Pat. No. 5,534,226 assigned to International Technidyne Corporation, discloses an apparatus and method for performing a coagulation time test on a blood sample whereby the blood is deposited into a capillary via a reservoir disposed within a disposable cuvette. The sample is then caused to reciprocally move within the capillary and blood forced to transverse a restricted region. Coagulation is determined to have occurred when the time required to transverse the restricted region is a predetermined percentage longer than the previous time.
U.S. Pat. No. 6,060,323 assigned to Hemosense, discloses a single use electronic device and test card for the measurement of the coagulation or lysis of a blood sample. The sample is caused to contact two electrodes, which measure the change in impedance corresponding to the change of viscosity of the sample as it clots.
U.S. Pat. No. 4,849,340, assigned to Cardiovascular Diagnostics, discloses an optical detection method for the determination of prothrombin time whereby capillary action is used to draw a predetermined volume of a liquid sample into a reaction chamber. Magnetic particles are caused to mix with the sample in a reaction chamber that are then agitated by an oscillating magnetic field. Light is shone onto the sample and subsequently detected. The point of coagulation is determined from the change in degree of the magnetic particle movement.
WO96/00390 discloses a fully disposable single-use device for determining blood-clotting activity whereby the distance traveled by the sample along a porous substrate is indicative of the clotting time.
U.S. Pat. 5, 039,617 assigned to Biotrack, describes a method and capillary flow device for carrying out measurement of Activated Partial Prothrombin Time (APTT) analysis on a capillary blood sample. The clotting time is measured by the cessation of blood flow in the capillary. The flow rate may be determined by flow or pressure sensors. The width of the capillary can vary from 0.05–3 mm and requires a sample volume of no more than 40 ul. Alternatively, if the sample contains particles, flow can be detected by observation of the speckle pattern resulting from the interaction of a light source, e.g. LED or laser, with the agitated particles in the capillary track.
The relationship between the changing impedance of a clotting blood sample has been studied (American Journal of Clinical Pathology 67:470–476, 1977). Measurement of the impedance of a blood sample over time was made, the resulting impedance curve representing the various processes in involved during clotting.
Measurement of Platelet Aggregation
Platelets are colourless cell fragments of about 2–4 um in diamter and are present in blood. Normal platelet counts range from 180,000–400,000/uL, however a platelet count of 50,000/uL is suffice for normal hemostasis. After vascular damage, for example after surgery, higher platelet counts are needed, sometimes in excess of 100,000/uL. The purpose of platelets is to repair gaps in the blood vessel wall by either adhering to themselves or to damaged tissue. When cells become damaged, they release certain chemicals which cause the platelets to change from a discoid to a spherical form and become sticky, known as the the aggregation-adhesion reaction. Platelets are thought to play an important role in the pathogenesis of isechemic heart disease; acute myocardial infarctions and unstable angina are clinical conditions associated with increased concentrations of certain platelet factors. Furthermore platelet dysfunction is one o the several major causes of bleeding after cardiopulmonary bypass. Platelets are also thought to contribute to the long-term process of atherogenesis by the release of growth factors and platelet function may also be influenced by high and low density lipoproteins. Thus screening for platelet function is an important and common hematological test.
Traditionally, this measurement was carried out on samples of platelet rich and platelet poor plasma, denoted respectively as PRP and PPP, using a Born aggregometer which measures the transmission of light through the sample.
U.S. Pat. No. 4,319,194 discloses an aggregometer which is able to carry out platelet analysis on whole blood. Wire shaped electrodes are inserted into the blood sample to which an aggregating agent is added and the change in impedance is recorded as a function of time. However movement in the wires causes variability in the interelectrode dictance and in the impedance measurements.
U.S. Pat. No. 6,004,818 asigned to Chrono-Log Corporation, discloses a method to measure platelet aggregation whereby the sample is caused to flow between adjacent parallel surfaces of electrode tips which define a channel. The electrodes are placed into a cuvette filled with the blood sample along with a means for stirring the sample.